Plant Science 155 2000 21 – 29
The maize streak virus coat protein transcription unit exhibits tissue-specific expression in transgenic rice
G. Mazithulela
1
, D. Sudhakar
2
, T. Heckel
3
, L. Mehlo, P. Christou, J.W. Davies, M.I. Boulton
John Innes Centre, Norwich Research Park, Colney, Norwich NR
4 7
UH, UK Received 20 October 1999; received in revised form 29 October 1999; accepted 3 December 1999
Abstract
Maize streak geminivirus MSV is a single-stranded DNA virus that infects cereals and other grasses. A promoter region incorporating the MSV large intergenic region and movement protein gene sequence was ligated to the gus b-glucuronidase
reporter gene which replaced the virus coat protein CP gene. The CP promoter activity was analysed in transgenic rice plants Oryza sati6a L. and was compared with that obtained in plants transformed with the gus gene downstream of the cauliflower
mosaic virus CaMV 35S promoter. The MSV CP promoter activity varied in the five plant lines tested, but was always less than that of the CaMV promoter. Histochemistry showed that the MSV CP promoter was active in cells of regenerating callus but in
regenerated plants it provided an expression pattern restricted to the vascular tissues of the root, stem, leaf and floral organs. Expression was highest in phloem-associated tissues of the vegetative organs and was absent from the tip and elongation region
of seedling roots. Thus, the MSV CP promoter shows a degree of developmental regulation and can be used to confer tissue-specific expression in transgenic rice plants. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
GUS; Tissue-specific expression; Maize streak virus; MSV; Coat protein promoter; Transgenic rice www.elsevier.comlocateplantsci
1. Introduction
The 35S promoter of cauliflower mosaic virus CaMV has been widely used to provide expres-
sion of foreign genes in transgenic plants. How- ever, there is a need for a wider range of
promoters with different characteristics, for exam- ple, to obtain different levels of transgene expres-
sion, expression at different developmental stages, or in specific organs and cell types. Furthermore,
although the CaMV promoter is suitable for ex- pression of foreign genes in transgenic rice, it
provides only low expression in other economi- cally important cereals and grasses of the family
Poaceae. Recent successes with the genetic trans- formation of maize [1 – 4], rice [5,6], wheat [7], oat
reviewed in [8], and barley [9] have required the identification of appropriate promoters for the
genetic modification of these crops.
Isolation of promoters from plants is ongoing but plant DNA virus promoters are now accessible
and, as was the case for CaMV, may contain cis regulatory elements able to confer specific patterns
of transgene expression in plants [10]. The pro- moters of other pararetroviruses such as cassava
vein mosaic virus CsVMV, [11], Commelina yel- low mottle virus CoYMV, [12], sugarcane bacilli-
form virus ScBV, [13] and rice tungro bacilliform virus RTBV, [14,15] have been investigated for
their ability to promote gene expression in trans- genic cereals. All but CsVMV naturally infect
monocots, but only ScBV and RTBV infect mem-
Corresponding author. Tel.: + 44-1603-452571; fax: + 44-1603- 456844.
E-mail address
:
margaret.boultonbbsrc.ac.uk M.I. Boulton
1
Present address: Dupont Agricultural Enterprise, Stine-Haskell Research Centre, P.O. Box 30, Newark, DE 19711, USA.
2
Present address: Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore 641003, India.
3
Present address: RDP, Ens-Lyon, 46 Allee d’Italie, F-69364 Lyon Cedex, France.
0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 2 5 6 - 3
bers of the Poaceae. The pattern of transgene expression, identified by b-glucuronidase GUS
staining, was not identical for the promoters of these viruses, with the CsVMV and ScBV pro-
moters being active in all organs tested and providing near constitutive expression, whereas
the RTBV and CoYMV promoters were specific to the vascular tissue. Only the RTBV promoter was
tested in its natural host species. Another plant DNA virus family, the Geminiviridae, contains a
genus Mastre6irus, [16] whose species infect members of the Poaceae. These viruses include
maize streak virus MSV, wheat dwarf virus WDV and Chloris striate mosaic virus CSMV.
Of these, MSV has the widest host range, infecting most of the economically important Poaceae [17].
However, the activity of the Mastre6irus pro- moters in transgenic cereals has not yet been
reported.
MSV possesses a single-stranded circular DNA genome but transcription of the double-stranded
viral DNA is bidirectional, initiating mainly in the large intergenic region LIR, and terminating in
the small intergenic region SIR which contains polyadenylation signals [18 – 20]. The LIR contains
the rightward promoter element rpe
1
[21] from which the virion sense genes encoding the move-
ment protein MP and coat protein CP are expressed [20].
To study the strength and tissue specificity of the MSV CP promoter region in transgenic rice, a
cp-gus reporter gene replacement construct was made. The construct contained the MSV LIR and
SIR, the mp gene sequence and parts of the repli- cation associated genes and therefore constituted
an MSV CP ‘extended promoter’. This MSV pro- moter conferred tissue specific expression and
showed some developmental regulation in trans- genic rice plants. The properties of this promoter
are compared with those of the CaMV 35S pro- moter, and other virus promoters, in the context
of their use for tissue-specific expression in trans- genic rice.
2. Methods
